GmAKT1-mediated K+ absorption positively modulates soybean salt tolerance by GmCBL9-GmCIPK6 complex

Abstract

Soybean is one of the most important crops in the world. However, salt stress poses a major challenge to soybean growth and productivity. Therefore, unravelling the complex mechanisms governing salt tolerance in soybean is imperative for molecular breeding of salt-tolerant varieties to improve yield. Maintaining intracellular Na⁺/K⁺ homeostasis is one of the key factors for plant salt tolerance. Although some salt tolerance mechanisms involving Na⁺ exclusion have been well identified in plants, few studies have been conducted on how K⁺ influx controls soybean salt tolerance. Here, we characterized the function of soybean K⁺ channel gene GmAKT1 and identified GmCBL9-GmCIPK6 complex, which modulated GmAKT1-mediated K⁺ uptake under salt stress. Functional studies found that soybean lines GmAKT1 overexpressing increased K⁺ content and promoted salt tolerance, while CRISPR/Cas9-mediated disruption of GmAKT1 soybean lines decreased the K⁺ content and showed salt sensitivity. Furthermore, we identified that GmCIPK6 interacted with GmAKT1 and GmCBL9 interacted with GmCIPK6. In addition, Mn²⁺-Phos-tag assays proved that GmCIPK6 could phosphorylate GmAKT1. This collaborative activation of the GmCBL9-GmCIPK6-GmAKT1 module promoted K⁺ influx and enhanced soybean salt tolerance. Our findings reveal a new molecular mechanism in soybeans under salt stress and provide insights for cultivating new salt-tolerant soybean varieties by molecular breeding.

Keywords: GmAKT1; GmCBL9; GmCIPK6; Salt stress; Soybean.

Figure 1

K⁺ uptake activity of GmAKT1 in yeast and Arabidopsis. (a) GmAKT1 improves the growth of K⁺ uptake‐deficient yeast mutant R5421 on the AP medium containing different K⁺ concentrations. The transformation of pYES2 vector as negative control. GmAKT1 was constructed into pYES2 vector and was transformed into yeast mutant R5421. The transgenic yeast cells were spotted in a 10‐fold dilution series on the different K⁺ concentrations medium. (b) Phenotypic analysis of WT, atakt1 mutant and Arabidopsis atakt1‐complemented lines (atakt1‐GmAKT1‐1 and atakt1‐GmAKT1‐2) under different K⁺ conditions for 16 days. (c) Fresh weight of WT, atakt1 and two atakt1‐complemented lines (atakt1‐GmAKT1‐1 and atakt1‐GmAKT1‐2) under various K⁺ concentrations. Each bar is shown as mean ± SD from n = 12 seedlings. Different letters indicate significant differences (P < 0.05, one‐way ANOVA). (d) Kinetics of K⁺ uptake activity among WT, atakt1 and two atakt1‐complemented lines by the depletion of K⁺ assay. The data represent three independent experiments.

Figure 2

GmAKT1 positively regulates salt resistance in soybean. (a) Phenotypic identification of different soybean lines (WT, GmAKT1‐OE lines and GmAKT1‐KO lines) under normal and salt stress (150 mM NaCl) for 32 days. Scale bar is 2 cm. (b‐e) The representative photographs of soybean lines Fv/Fm in leaves (b), Ion leakage from leaves (c), fresh weight of roots (d) and fresh weight of leaves (e). (f–k) The values of Na⁺ (f–g), K⁺ (h–i) and Na⁺/K⁺ ratio (j–k) of roots and leaves were shown in different soybean plants. For each assay, the data are shown as mean ± SD, n = three biologically independent experiments. Lowercase letters indicate significant differences between samples at P < 0.05, according to two‐way ANOVA.

Figure 3

The interaction analysis between GmAKT1 and GmCIPK family members. (a) Yeast two‐hybrid assays (Y2H) between the C‐terminal part of GmAKT1 (GmAKT1‐C) and the full length 26 GmCIPK family members. pGBKT7‐53 + pGADT7‐T serves as positive control, pGBKT7‐Lam + pGADT7‐T and GmAKT1‐C + pGADT7‐T represent negative control. The 1:10 serial dilutions of yeast cells were spotted on synthetic defined (SD) medium lacking Leu and Trp (SD‐TL) or SD‐Leu‐Trp‐His‐Ade (SD‐TLHA) with X‐α‐gal medium, respectively. (b) Bimolecular fluorescence complementation (BiFC) analysis for GmAKT1 and GmCIPK6 interaction in tobacco leaves. The YFP fluorescence was obtained by confocal microscopy. GmAKT1‐nYFP+cYFP and nYFP+GmCIPK6‐cYFP as negative control and GmAKT1‐nYFP+GmCIPK6‐cYFP as interaction proteins. PM‐RFP as a cell membrane marker. Scale bar is 50 μM. (c) Co‐IP assays showing that GmAKT1 interacts with GmCIPK6. Co‐IP assay showing that GmAKT1 interacts with GmCIPK6 in tobacco leaves. Total proteins were immunoprecipitated with anti‐HA antibody (IP: HA) and were identified with anti‐LUC antibody (IB:LUC). IgG antibody as a negative control. (d) GmCIPK6 interacts with GmAKT1 domain in yeast cells. Yeast cells were grown on synthetic defined SD‐TL medium and SD‐TLHA medium. For each experiment, three independent experiments were repeated with similar results.

Figure 4

GmAKT1 is phosphorylated by GmCIPK6. (a, b) Western blot showing the phosphorylation of GmAKT1 in soybean with Mn²⁺‐Phos‐tag SDS‐PAGE. SDS‐PAGE as control for GmAKT1‐HA expression. Protein extracts were treated with or without calf intestinal phosphatase (CIP) and immunoblot detected with anti‐HA antibody. (c) Purified recombinant protein GmAKT1‐c‐MBP, GmAKT1‐c^S730A‐MBP and GmCIPK6‐His were incubated together and detected by western blot with SDS‐PAGE and Mn²⁺‐Phos‐tag SDS‐PAGE. All experiments included three biological replicates.

Figure 5

GmCIPK6 and GmCBL9 interact with each other. (a) Identifying interactions between GmCIPK6 and GmCBL proteins via yeast two‐hybrid (Y2H) assay. Transformed yeast cells were grown on SD medium (lacking Leu and Trp) or (lacking Leu, Trp, His and Ade). The combination pGBKT7‐53 and pGADT7‐T was used as a positive control, and pGBKT7‐lam + pGADT7‐T and GmCIPK6+ pGADT7‐T were used as a negative control. (b) Bimolecular fluorescence complementation (BiFC) reveals the interaction between GmCIPK6 and GmCBL9 in tobacco leaves. The N‐ and C‐terminal fragments of YFP were linked to the GmCBL9 and GmCIPK6, respectively. GmCBL9‐nYFP+cYFP and nYFP+GmCIPK6‐cYFP were used as negative control. PM‐RFP as a cell membrane marker. Scale bars are 50 μM. (c) Co‐IP assay showing the interaction between GmCIPK6 and GmCBL9. GmCBL9‐FLAG and GmCIPK6‐LUC were expressed in tobacco and then used for Co‐IP assays. Anti‐FLAG and anti‐LUC antibodies were used to detect GmCBL9‐FLAG and GmCIPK6‐LUC, respectively. Three independent experiments were performed in this study.

Figure 6

Coexpression of GmCBL9 and GmCIPK6 enhances GmAKT1 medicated K⁺‐uptake activity. (a–c) GmCBL9‐GmCIPK6 improves the growth of K⁺‐uptake‐deficient yeast mutant R5421 expressing GmAKT1 on different K⁺ AP medium. (d) The GmAKT1‐mediated regulation of K⁺ involving GmCBL9 and GmCIPK6 enhances yeast growth on a salt medium. GmAKT1, GmCBL9 and GmCIPK6 were constructed into p416 and p424 vectors, respectively. The 1:10 serial dilutions (as a black triangle) of yeast cells were spotted on the AP medium.

Figure 7

GmAKT1‐mediated K⁺ inward currents are activated by GmCBL9 and GmCIPK6 in HEK293 cells. (a–d) Patch‐clamp whole‐cell recordings of inward K⁺ currents in HEK293 cells expressing different combinations of GmAKT1, GmCBL9‐GmCIPK6‐GmAKT1, GmCBL9‐GmCIPK6‐GmAKT1^S730A and GmCBL9^E172Q‐GmCIPK6‐GmAKT1. The voltage protocols, as well as time and current scale bars for the recordings, are shown. (e) The I‐V relationship of the steady state whole‐cell inward K⁺ currents in HEK293 cells. The data are derived from the recordings as shown in (a–d) and presented as means ± SE.

Figure 8

Soybean K⁺ channel GmAKT1 enhances salt‐tolerant capacity by the regulation of GmCBL9‐GmCIPK6 complex. (a) Soybean plant phenotypes under normal and salt stress conditions. Bars, 2 cm. GmCBL9‐GmCIPK6‐OE/GmAKT1‐KO hairy roots in the GmAKT1‐KO background, GmCBL9‐GmCIPK6‐OE/WT hairy roots in the WT (DN50) background, and GmCBL9‐GmCIPK6‐OE/GmAKT1‐OE hairy roots in the GmAKT1‐OE background. Data are the mean ± SD of n = 3. (b and d) Na⁺ content, K⁺ content and Na⁺/K⁺ ratio of GmCBL9‐GmCIPK6‐OE/GmAKT1‐KO, GmCBL9‐GmCIPK6‐OE/WT and GmCBL9‐GmCIPK6‐OE/GmAKT1‐OE hairy roots under normal and 150 mM salt stress. n ≥ 30 plants per genotype. Different letters indicate statistically significant differences at P < 0.05 by two‐way ANOVA and Student's t‐test.

Figure 9

A proposed model for GmCBL9‐GmCIPK6‐GmAKT1 module medicated salt stress response in soybean. Under salt stress, Ca²⁺ sensor GmCBL9 interacts with protein kinase GmCIPK6 and recruits GmCIPK6 to the plasma membrane where they phosphorylate and activate GmAKT1‐mediated K⁺ uptake, thus keeping Na⁺/K⁺ ratio and enhancing salt tolerance of soybean.